Insulin action at the cellular level is initiated by binding of the hormone to a specific site of the surface membrane of target cells termed a receptor. This receptor is a high molecular weight glycoprotein with two major subunits. In this proposal, we will study directly the structure of the insulin receptor and its synthesis and degradation in cultured cells and intact animals. Receptors will be labelled both biosynthetically and using surface labeling techniques. Biosynthetically both 35S-methionine and 3H-sugars will be used. Surface labeling of protein will be achieed with lactoperoxidase induced iodination and terminal sugars will be labelle by NaB3H4 reduction after either galactose oxidase or periodate oxidation. The insulin receptor will be identified by immunoprecipitation with antireceptor antibodies and gel electrophoresis. The structure of the receptor will be studies in both functional (hepatoma) and non-functional (IM-9 lymphocyte) cells. The structure of receptors on plasma membranes and intracellular organelles will be compared in both reduced and non-reduced gels. A third subunit of 206K has been identified which will be characterized by peptide mappking, and the stoichiometry of the receptor subunits determined. The transmembrane nature of the receptor and possible modes of transmembrane signaling will be investigated, and the role of receptor glycosylation determined in both structure and function. The synthesis and degradation of insulin receptors will b studies after both biosynthetic labeling and external labeling of cells. The normal rates of synthesis and degrdation and the effects of various hormones and drugs on these rates will be evaluated. We will also be able to elucidate the biosynthetic and degradative pathways, and study the mechanism by which insulin regulates its own receptor. tudies in intact animals with both diabetes and obesity will also be conducted and the effect of insulin treatment and diet on receptor turnover examined. These studies will provide basic information about the mechanism of insulin action, and in addition, new strategies in the treatment of both insulin-dependent and non-insulin dependent diabetes mellitus.